Report for 9/4/2013 through 9/2/14: Organization and Infrastructure This year Dr. Feldman Instituted a system of monthly charges to NIH Z-core users, as a mechanism for partial cost recovery, which began in November 2013. Implemented formal monthly lab meetings with interested PIs from client labs. This is currently done with the Stratakis and Kaler laboratories. Took a leading role on the acquisition, installation, troubleshooting and protocol establishment of a state-of-art suite of equipment designed to monitor behavior of zebrafish embryos, larvae, juveniles and adults. This equipment from ViewPoint Life Sciences was jointly purchased by NICHD and NHGRI. Created one online calendar to reserve microscopes and equipment in the Z-core & Shared Imaging Room & one online calendar to reserve use of the WT EK breeder fish he maintains in B134 for Z-core users. Reorganized the layout of equipment in B140, preserving all research functionalities, while increasing usable bench space. Increased the Z-cores capacity for storing zebrafish embryos with the acquisition of a new incubator. Acquired six DC-96 multiplex zebrafish aquaria to help Z-core users more efficiently manage their genotyping and other scenarios in which multiple zebrafish need to be individually housed. New Research Projects and Completed Research Projects A new project was established with Dr. Kandice Tanner from NCI aimed at measuring human tumor cell invasiveness using zebrafish embryonic and larval tissues as the biological substrate. Projects with the DeCherney lab and the Lenardo lab were brought to a conclusion; it is not clear whether the data they acquired will be published. Projects with Dr. Weis (NIEHS) have remained inactive. Summary of Research, Training and Consultation Research Category 1: TALEN and CRISPR/Cas9-based genome editing for zebrafish gene knockouts. TALEN alleles for the Stratakis lab have been outcrossed in anticipation of documenting the homozygous recessive phenotype. The Porter lab has generated a substantial body of solid data using TALEN alleles that indicate female zebrafish-specific requirements for sterol homeostasis among other phenomena. The CRISPR/Cas9 system has brought huge decreases in cost and complexity of creating mutant alleles. As such, many NIH labs now use the system independently. Dr. Feldman has nonetheless continued to stay on top of progress and innovations in the field and at NIH to purchase and stock relevant new DNA constructs of interest and to compile useful protocols. Research Category 2: Medium-throughput drug screening in zebrafish. With assistance from the Core and their K01 grant, Dr. Ljuba Caldovic and the Tuchman lab from Childrens National Medical Center continue in their effort to identify neuroprotectants against hyperammonemia in a small molecule screen using zebrafish. A paper was published this year and substantive improvements have been subsequently made leading to the identification of several lead compounds from a library of FDA-approved small molecules. These improvements include 1) delaying the placement of embryos into 96-well plates until the day of the screen and 2) using the ViewPoint system to monitor changes in activity, with non-activity serving as proxy for neurotoxicity-induced death. Research Category 3: Phenotyping Genetic Animal Models. The Stratakis lab is interested in several gene classes. Class A show an association of debilitating mutations with human adrenal hyperplasia. Class B show an association of activating mutations. Class C show an association of duplication with gigantism. Progress this year for one Class A genes has involved the generation of a knockout allele in the zebrafish orthologue and the recovery of this allele after near-loss due to husbandry challenges. For the other Class A gene, a potential RNAseq-based research paradigm for rapid functional insights has been explored. Transient loss-of-function (LOF) and gain-of-function (GOF) embryos were generated by microinjecting, respectively, an antisense morpholino oligonucleotide that targets a zebrafish orthologue or in vitro-synthesized RNA encoding this orthologue. Whole RNA was harvested from these embryos, together with RNA from control embryos and RNAseq was outsourced. Ongoing analysis of the data suggests that a focus on genes reciprocally regulated by LOF and GOF perturbations can help avoid artifacts often associated with transient LOF or GOF alone. Through this work, Dr. Feldman has acquired expertise on the bioinformatic analysis of raw RNAseq data and an expansion of the Z-cores capacity for such studies is planned. For the Class B genes a strategy for transient GOF via DNA-based transgenes has been laid out and the contributing DNAs have been collected. Projects for both Class A and Class B genes will require visualizing changes to the zebrafish equivalent of the adrenal gland: the interrenal primordium (IRRP). To this end whole-mount in situ hybridization (WISH) probes have been assembled and two of them are workinf well for visualizing the IRRP. For functional studies of Class C genes, we have obtained a set of pituitary and hypothalamus WISH probes for which the pituitary ones are working well. Our first goal is to determine the effect of GOF (via RNA injection) of zebrafish orthologues to Class C genes on pituitary and hypothalamus size and function. Our second, and related goal is to learn how Class C orthologue GOF affects the size and growth rate of fish. For this, it will likely be necessary to introduce DNA-based transgenes rather than RNA and a strategy for their construction has been established. We also investigated the possibility of a growth effect from RNA injection alone. We cannot conclude whether or not there is an effect of RNA GOF on growth, but through endeavors we developed new tool for size measurement and an improved experimental design for such studies. For all Class A, B and C genes, we plan to document the native expression pattern of the relevant zebrafish orthologues. We have partially completed this task for one of the Class A genes. For the Kaler lab studies, we intend to document motor neuron development in an established fish model that is mutated for the orthologue to their disease gene of interest. This year we obtained such fish, as well as a transgenic line of fish that expresses green fluorescent protein (GFP) in motor neurons. We have intercrossed these two lines of fish to generate founders that when incrossed will yield mutant progeny with GFP-labeled motor neurons. In addition to visualizing motor neurons by fluorescence of GFP, we plan to use antibodies against GFP or against native motor neurons (the Zn5 antibody) for rapidly assessing larger cohorts of progeny. We have added an antibody-specific program to the Z-cores semi-automated incubation system that is already successfully used for WISH. The second goal of this project is to determine whether different alleles of the human form of this gene show different abilities to rescue motor neuron defects. We have devised a set of constructs to achieve this via synthetic RNA injection, and molecular cloning is underway. Molecular cloning is also underway to create constructs for synthesizing WISH probes that will enable us to examine the native expression profile of the zebrafish orthologue. Training and Consultation. The relative experience working with the zebrafish model of the individuals conducting the above research varies considerably, but in all cases Dr. Feldman is heavily involved in consultation and/or hands-on training. Hands-on training has been particularly required for those researchers from the Stratakis and Kaler labs who began their research this year with no zebrafish research experience at all.